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Original article A genetic analysis of variable number of tandem repeats (VNTR) polymorphism in the horse G Guérin M Bertaud B Billoud JC Mériaux 2 1 INRA, Laboratoire de Génétique Biochimique; 2 INRA, Laboratoire des Groupes Sanguins, 78352 Jouy-en-Josas Cedex, France (Received 3 December 1992; accepted 1 July 1993) Summary - Restriction fragment length polymorphism detected with the 33.6 minisatel- lite probe (Jeffreys et al, 1985) was analysed in 3 horse families of paternal half-sibs and in a sample of stallions from 4 breeds. Among the bands detected on Hae III genomic DNA digests, it was found that strongly hybridizing fragments behaved as alleles at 2 different loci. These 2 loci, showing 7 and 3 detectable alleles, were not closely linked to each other nor to informative blood groups and protein markers. No neo-mutation in allele length was observed at these 2 loci ip the 3 families. In the stallion sample, 8 alleles were detected at the first locus and no differences were found between their frequencies in the 4 breeds. Heterozygosity and polymorphism information content (PIC) values estimated in the 4 breeds show that the Thoroughbred is the least variable breed and the Arab the most. In the whole population the PIC values were 0.73 and 0.70 respectively. horse / variable number of tandem repeats / linkage / genetic marker / polymorphism Résumé - Analyse génétique du polymorphisme du nombre variable de répétitions en tandem (VNTR) chez le cheval. Le polymorphisme de longueur de fragment de restriction détecté par la sonde minisatellite .!,!.6 (Jeffreys et al, 1985) a été analysé chez le cheval dans trois familles de demi-germains paternels et dans un échantillon d’étalons de quatre races. Parmi toutes les bandes révélées à partir d’ADN génomique digéré par HaeIII, les fragments donnant un signal intense se comportent comme des allèles ségrégeant à deux locus différents. Ceux-ci, qui possèdent respectivement sept et trois allèles détectables, ne sont pas étroitement liés entre eux ni avec les locus informatifs des groupes sanguins et des protéines du sang. Aucune néo-mutation de longueur des allèles n’a été détectée dans les trois familles. Dans l’échantillon des étalons, huit allèles ont été observés au premier locus et aucune différence de fréquence n’apparaît entre les quatre races. Les valeurs d’hétérozygotie et les valeurs informatives des polymorphismes (PICJ estimées sur cet échantillon montrent que la race de Pur-sang est la moins variable à l’opposé de la race Arabe. Les valeurs estimées sur la population totale sont respectivement de 0,73 et de 0, 70. cheval / nombre variable de répétitions en tandem / liaison / marqueur génétique / polymorphisme INTRODUCTION It is now well established that the genome of a number of animal and plant species contains regions that are highly variable, among which are the minisatellites. These regions are detected by probes from a different origin containing sequences several base-pairs long, tandemly repeated. Initially, highly polymorphic patterns displaying a large number of fragments on Southern blots resulting from variation in the number of tandem repeats and called fingerprints were obtained in man by Jeffreys et al (1985). These authors used a repeat found in the second intron of the myoglobin gene to design a probe that revealed related human sequences, some of which were themselves later used as probes. This was the case for the 33.6 and 33.15 regions that revealed extensive polymorphism in a number of organisms. Other probes containing repeated sequences such as a sequence from the M13 bacteriophage (Vassart et al, 1987), a sequence homologous to the Drosophila Per gene (Shin et al, 1985), in the 3’ region of the a globin gene (Jarman et al, 1986) and a number of others, cloned from the human genome (see for example Nakamura et al, 1987), were found to detect very high levels of polymorphism. In pets and domestic farm animals, polymorphisms were found in dogs and cats (Jeffreys and Morton 1987), in cattle (Georges et al, 1988), in poultry (Hillel et al, 1989), in sheep (Drinkwater et al, 1990) and in pigs (Coppieters et al, 1990) using either human, viral or homologous probes. In horses, restriction fragment length polymorphisms (RFLP) have been detected using different probes such as M13, Jeffreys’ minisatellites or core sequence, Per, human VNTR, (TG)n, a homologous horse probe and synthetic tandem repeats (Georges et al, 1988; Massina et al, 1989; Troyer et al, 1989; Bernoco and Byrns, 1991; Broad et al, 1991; Hopkins et al, 1991; Ellegren et al, 1992; Giulotto et al, 1992; Mariat et al, 1992). However, very little or no information about segregation of the fragments and linkage analysis has been reported as limited families were typed. In this report, we show the segregation of DNA fragments hybridized with a human minisatellite probe in 3 large horse pedigrees and give some genetic characteristics of the regions detected in 4 horse breeds. MATERIALS AND METHODS Animals Three horse families consisting of a stallion and half or full paternal sibs, along with their mothers, were collected. The number of mother (74) - offspring (87) combinations were 30, 27 and 30 for the 3 stallions, Iris Landais, Artichaut and Matador du Bois, respectively. All animals were of Selle Fran!ais origin. A sample of stallions from 4 breeds, Arabs (AR, N = 47), Thoroughbred (TB, N = 43), Selle Fran!ais (SF, N = 40) and Trotteur Fran!ais (TF, N = 43) were also obtained from different state stud-farms. All animals were blood typed by the Laboratoire des Groupes Sanguins des Chevaux, INRA-CRJ, Jouy-en-Josas, for the blood group systems (A, C, D, K, P, Q, U) and the protein systems (Alb, Tf, A1B, Es, Pi, Gpi, Pgd, Pgm) and all offspring qualified. Purification of DNA Blood samples were drawn in tubes containing tripotassium EDTA (Vacutainer) and DNA was extracted according to the method of Jeanpierre (1987). Blood was treated twice in a solution of 10 mM NaCI, 10 mM EDTA, pH 8, for 30 min at +4°C. Pellets containing leucocytes were homogenized in a solution of 5 M guanidine HCI, 0.5 M ammonium acetate, 1.25% N-lauroylsarcosine containing 0.1 mg/ml proteinase K for 1 h at 60°C. DNA was then precipitated with 2 vol ethanol and washed twice with 70% ethanol. Restriction endonuclease digestion and electrophoresis Seven pg samples of horse DNA were digested for 20 h at 37°C by restriction endonucleases HaeIII or HinfI (BRL or Boehringer) according to the manufacturer’s recommendations. Complete digestion was achieved with a total addition of 5 units per Ag DNA. Restriction endonuclease DNA fragments were subjected to horizontal electrophoresis in 1% agarose gels 25 cm in length in Tris-acetate buffer (40 mM Tris, 20 mM AcOH, 2 mM EDTA; pH 8.3). Electrophoresis was performed with circulating buffer at 55 V (HaeIII) or 60 V (Hinfl) for 18 h. Calibration was achieved by adding a sample of BstEII-KpnI-SmaI digested lambda phage DNA. Transfer Transfer was performed on Biodyne B membrane (Pall Industry, SA). The DNA digested fragments in the gels were depurinated in 0.15 N HCI for 10 min, denatured in a 0.4 N NaOH solution for 30 min and transferred using the method of Southern (1975) at room temperature for 18 h with 0.4 N NaOH. Hybridization probes The 2 human VNTR probes, 33.6 and 33.15 were kindly provided by AJ Jeffreys. They were labelled by the primer extension method according to Jeffreys (1985), except that the radiolabelled insert was not extracted from the plasmid. Prehybridization and hybridization Prehybridization was carried out for at least 4 h at 65°C in a mixture containing 6 x SSC (0.09 M trisodium citrate, 0.9 M NaCI), 0.25% skimmed milk, 0.5% Denhardt’s solution, 0.5% SDS). Hybridization was performed in the same solution except that 0.05% sodium pyrophosphate was added and sufficient radiolabelled probe to provide !! 1 x 10 6 cpm/ml. Membranes were then washed twice for 15 min at room temperature in 2 x SSC, 0.1% SDS then in the same solution for 30 min at 65°C (in more stringent solution at the same temperature if necessary) and finally rinsed in 2 x SSC at room temperature. The restriction endonuclease fragments were vizualized by autoradiography in a cassette with a Kodak XAR film. and Cronex LI-PLUS intensifying screens at -70°C. Dehybridization This was performed by washing the blot for 1 h at 37°C with 0.4 N NaOH, followed by washing for 1 h in a 0.1 x SSPE, 0.5% SDS, 0.2 M Tris-HCI pH 7.5 solution (1 x SSPE = 0.18 M NaCI, 0.01 M NaH2P0!, 1 mM EDTA). Linkage analysas Calculations were perforined according to the Lod score method of Morton (1955). RESULTS Probes and enzymes DNA extracted from our group of horses was digested with HaeIII and with Hinfl. Whatever the enzyme used, the 33.15 probe gave a large number of fragments with rather homogeneous intensities and no clear polymorphism (results not shown). Conversely, the 33.6 probe detected fewer bands with different hybridization signal intensities. With Hinfl, all strongly hybridizing bands were detected in the range of small fragment lengths (less than 3.5 kb) (fig la). HaellI gave more evenly distributed bands spread over the autoradiograms (fig lb). Since, with DNA digested with Hinfl, the different levels of band migration appeared to be more heterogeneous than with HaeIII, it was decided to take into account only results obtained with the 33.6-HaeIII probe-enzyme combination. Fragment identification and polymorphism Blots usually consisted of DNA digests from 1 sire and its offspring along with DNA digests from their mothers. The 3 sires were also run on the same gel to compare the respective mobilities of their bands (fig 2). Two major groups of bands with a strong radioactive signal appeared in the range of medium (2.5 to 5 kb) and small fragment length inferior to 2 kb (fig 2) on HaeIII blots probed with 33.6. A total of 15 fragments were given a letter as an identification code (A to 0) according to their mobility and intensity while the rest of the bands giving low signals were not included in our interpretation. All strongly hybridizing fragments were found to be polymorphic. 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In the whole population the PIC values were 0.73 and 0.70 respectively. horse / variable number of tandem repeats / linkage / genetic marker / polymorphism Résumé. long, tandemly repeated. Initially, highly polymorphic patterns displaying a large number of fragments on Southern blots resulting from variation in the number of tandem repeats